Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis.
After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
von Wintzingerode
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Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Berlin, Germany.
Göbel
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UB
Stackebrandt
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D016477:Artifacts; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004783:Environmental Microbiology; D004866:Equipment Contamination; D005188:False Negative Reactions; D005189:False Positive Reactions; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S